Many of the molecules that are critical for the detection and transduction of odors by olfactory sensory neurons (OSNs) have been identified, and their basic roles in these processes defined. However, significant gaps in our understanding of the olfactory transduction process remain. For example, signal transduction cascades found in many neurons function within signaling complexes composed of receptors, effector enzymes, channels, scaffolding elements and other molecules. These signalplexes may enhance both the efficiency and specificity of signaling by increasing the local concentration of signaling elements (e.g., receptors, enzymes and soluble messengers such as cAMP or Ca2+), restricting proteins to functionally important cellular domains (e.g., dendritic spines or dendritic cilia), orby regulating access of modulatory proteins (e.g., receptor kinases, -arrestin). However, little is known about how olfactory transduction proteins interact to impact olfactory function. Our proposed studies will address this important yet understudied area of olfactory biology by using a cutting-edge, multidisciplinary approach to define protein-protein interactions for key components of the olfactory transduction cascade. Our studies will focus on identifying proteins that directly interact with two key olfactory transduction molecules: olfactory marker protein (OMP) and canonical odorant receptors (OR). While both OMP and the ORs have been shown to interact with other OSN proteins (Bex proteins in the case of OMP and receptor trafficking protein (RTP) family members in the case of ORs), functional studies suggests that other partners exist for both proteins. For example, OMP influences cAMP kinetics and Ca2+ dynamics in OSNs, while native ORs are hypothesized to require additional OSN-specific co-receptors or chaperones to efficiently target the plasma membrane. Recent advances in proteomics now offer a unique opportunity to both validate previously implicated OMP and OR interactors as well as to identify novel proteins that associate with these key transduction molecules. In this proposal, the P.I.s will take advantage of our complementary expertise in olfactory transduction and state-of-the-art proteomics approaches to complete two parallel Specific Aims focused on identifying protein interactors for two baits: OMP (Aim 1) and the heptanal-responsive OR I7 (Aim 2). We will use stable isotope labeling of mice expressing different levels of the either bait, followed by immunoprecipitation of interacting complexes from native olfactory epithelium and subsequent liquid chromatography-tandem mass spectroscopy to quantitatively isolate specific interactors with high sensitivity. These studies will result in significant advances in our understanding of odor detection and transduction, and will establish an important new approach for characterizing the interactions of rare proteins in nearly any biological system. PUBLIC HEALTH RELEVANCE: The reduction or loss of olfactory function due to injury, disease or aging can have serious consequences for health and quality of life, including appetite dysregulation, excessive consumption of sugary, fatty or salty foods, a decreased ability to avoid spoiled foods or to maintain proper hygiene, and an increased incidence of depression and social withdrawal. Furthermore, normal olfactory function can contribute to dysfunctional overconsumption and lead to the development of associated diseases, including obesity, diabetes, hypertension and cardiovascular disease. As both normal and abnormal olfactory function can contribute to life-threatening disease and reduce quality of life, it is imperative tht we better understand this critical sense; such understanding could lead to new interventions, including changes in processed foods, that would enhance human health.